1. Field
The invention relates to chimeric polypeptides that are chemically linked via cysteine-based disulfide bridges (e.g. cystine), methods for producing such polypeptides, and uses thereof.
2. Description
Artificial bifunctional or multifunctional biologically active compounds can be used in diagnostics and therapy, for example immunodiagnostics and immunotherapy. The specific binding of an antibody, or of an antibody fragment, to its antigen can advantageously be used to direct a protein having a different biological function towards the specific antigen. For instance, a bispecific antibody whose antigens are located, on the one hand, on tumor cells, and on the other hand, on macrophages, can be utilized for directing killer cells towards a tumor (Bohlen, H. et al., Blood 82 (1993) 1803-1812). Such bispecific antibodies can be produced by fusing two hybridoma cells that produce the respective monospecific antibodies, to form quadroma cells that may also produce bispecific antibodies (Milstein, C. and Cuello, A. C., Nature 305 (1983) 537-540). Regrettably, this method of obtaining bispecific proteins is limited exclusively to antibodies and only about 15% of those antibodies expressed exhibit the desired bispecificity. Furthermore, these antibodies have to be isolated by labor-intensive purification methods.
Another method of producing bispecific proteins is based on the chemical cross-linking of two proteins having the desired properties (Fanger, M. W. et al., Crit. Rev. Immunol. 12 (1992) 101-24). Cross-linking is accomplished by means of bifunctional linker molecules which react with amino groups of the proteins or with cysteine residues. In the latter case, cysteine residues of the one protein may be activated by 5,5xe2x80x2-dithiobis-(2-nitrobenzoic acid) (xe2x80x9cDTNBxe2x80x9d). The addition of the second protein which contains cysteine residues in reduced form causes the formation of disulfides, thereby covalently coupling the two proteins. Using this method, the yield of heterodimeric bifunctional proteins can be improved compared to non-specific cross-linking, which usually results in a high proportion of homodimers. Unfortunately, this method still results in non-homogeneous material that may impact negatively on the stability and functionality of the bispecific construct (Debinski, W. and Pastan, I., Bioconjug. Chem . 5 (1994) 40-43).
A common method for producing bifunctional proteins forms a DNA construct that links the 5xe2x80x2 end of a cDNA encoding a protein with the 3xe2x80x2 end of a gene encoding another protein, while retaining the reading frame. This DNA construct is then expressed recombinantly. In this manner, an antibody fragment directed against a tumor was fused with a bacterial toxin capable of specifically killing tumor cells (Brinkmann, U. et al., Proc. Natl. Acad. Sci. USA 88 (1991) 8616-8620). Fusions of antibody fragments were also successfully produced with RNase and other enzymes, and their functionality was examined in cell cultures (Newton, D. L et al., J. Biol. Chem. 267 (1992) 19572-19578; Zewe, M. et al., Immunotechnol. 3 (1997) 127-136).
Diabodies represent another form of fusion proteins (Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448). Diabodies consist of two Fv fragments having different specificities. Unlike scFv fragments in which the two variable domains of an antibody are connected to one another via a linker, in diabodies the VL domain of the one antibody is fused with the VH domain of a second antibody. The structure of the linker used for this purpose prevents intramolecular association of the two domains, and instead causes an intermolecular association of the two constructs, resulting in the formation of a bifunctional diabody.
Numerous attempts have been made to develop systems to produce bifunctional proteins. To this end, proteins were fused at the gene level with peptides or proteins as dimerization domains to impart directed association. As dimerization units were used the antibody domains CL and CHl, calmodulin and the corresponding binding peptide or streptavidine (Mxc3xcller, K. M. et al., FEBS Lett. 422 (1998) 259-264; Neri, D. et al., BioTechnology 13 (1995) 373-377; Dxc3xcbel, S et al., J. Immunol. Methods 178 (1995) 201-209). In addition, short peptide sequences such as leucine zippers and amphiphilic helices could also be used as functional units for the directed heterodimerization (Kostelny, S. A. et al., J. Immunol. 148 (1992) 1547-1553). However, to date, no generally applicable method for directed association has been established.
The subject invention provides a method for producing a chimeric polypeptide having two polypeptide chains that are linked to each other by 1 to 3 cysteine-based disulfide bridges. This method comprises providing a first polypeptide chain and a second polypeptide chain, bringing the first polypeptide chain into spacial proximity with the second polypeptide chain under conditions such that the basic amino acids on the first polypeptide chain interact ionically with the acidic amino acids on the second polypeptide chain, and treating the first polypeptide chain and the second polypeptide chain that have interacted ionically with each other with an oxidizing agent. In this method, the first polypeptide chain consists essentially of from 1 to 3 cysteines and from 4 to 12 basic amino acids selected from the group consisting of arginine, lysine, and ornithine. The first polypeptide has attached to its C- or N-terminus a first biologically active compound. The second polypeptide chain consists essentially of 1 to 3 cysteines and 4 to 12 acidic amino acids selected from the group consisting of glutamate and aspartate. The second polypeptide has attached to its C- or N-terminus a second biologically active compound.
With respect to the treating the first polypeptide chain and the second polypeptide chain that have interacted ionically with each other with an oxidizing agent, such oxidation is under conditions such that the 1 to 3 cysteines of the the first polypeptide chain form disulfide bridges with the 1 to 3 cysteines of the second polypeptide chain and thus produce the chimeric polypeptide. This chimeric polypeptide can then be isolated.
It is preferred that the first polypeptide chain and the second polypeptide chain each have 2 or 3 cysteines. The distance between any two cysteines in a polypeptide chain is preferably more than one amino acid, for example, from 3 to 6 amino acids.